This project is designed to examine the ways in which the heme moiety is able to activate small molecules, particularly O2 and H2O2, and utilize them in catalytic processes. The effects of specific iron coordination and axial ligation, heme and covalently modified heme electronic structure, and heme environment on chemical reactivity and specificity will be studied. 1H and 2H nuclear magnetic resonance (NMR) spectroscopic studies are a prominent feature of this project. All of the common spin, oxidation, and ligation states of iron (except low spin Fe(II)) are paramagnetic, and the unique features of paramagnetic NMR produce a probe that is effective in highlighting protons in the vicinity of the iron atom in hemes and the information gained from studies of model complexes is required for the interpretation of the NMR spectra of heme proteins. NMR studies will be conducted under carefully controlled conditions (particularly at low temperature) to detect reactive intermediates as they are formed during reactions with O2 and H2O2 in model compounds and in collaborative projects with heme proteins. The process of activation of small molecules frequently results in covalent modification of the heme. This project has recently discovered that iron porphyrins with a single meso substituent are remarkably sensitive to attack on the heme by O2. In order to understand the structure, reactivity and function of these modified hemes, studies of model complexes will be conducted by NMR techniques, X-ray crystallography, and a range of other spectroscopic techniques in order to provide benchmark compounds that allows us to understand the nature of the electronic and structural distortions that result from covalent modifications found in key intermediates in heme degradation, hydroxylated hemes, biliverdin complexes, etc., and which also enables us to identify these species as they are formed during chemical and enzymatic reactions. Covalent heme modification is particularly apparent in heme oxygenase where the heme, as substrate, undergoes regiospecific oxidation at the alpha-meso position and subsequent ring opening. We plan studies designed to address the issue of this regiospecificity through study of suitable model compounds and proteins. The nature of the important carbon-carbon bond breaking steps in heme degradation, which cannot be directly observed in proteins such as heme oxygenase, will be studied through the identification of reactive intermediates in model systems, particularly in highly reactive hemes and in sterically protected heme environments.